Influence of nanoparticle encapsulation and encoding on the surface chemistry of polymer carrier beads

Surface-functionalized polymer beads encoded with molecular luminophores and nanocrystalline emitters such as semiconductor nanocrystals, often referred to as quantum dots (QDs), or magnetic nanoparticles are broadly used in the life sciences as reporters and carrier beads. Many of these applications require a profound knowledge of the chemical nature and total number of their surface functional groups (FGs), that control bead charge, colloidal stability, hydrophobicity, and the interaction with the environment and biological systems. For bioanalytical applications, also the number of groups accessible for the subsequent functionalization with, e.g., biomolecules or targeting ligands is relevant. In this study, we explore the influence of QD encoding on the amount of carboxylic acid (COOH) surface FGs of 2 µm polystyrene microparticles (PSMPs). This is done for frequently employed oleic acid and oleylamine stabilized, luminescent core/shell CdSe QDs and two commonly used encoding procedures. This included QD addition during bead formation by a thermally induced polymerization reaction and a post synthetic swelling procedure. The accessible number of COOH groups on the surface of QD-encoded and pristine beads was quantified by two colorimetric assays, utilizing differently sized reporters and electrostatic and covalent interactions. The results were compared to the total number of FGs obtained by a conductometric titration and Fourier transform infrared spectroscopy (FTIR). In addition, a comparison of the impact of QD and dye encoding on the bead surface chemistry was performed. Our results demonstrate the influence of QD encoding and the QD-encoding strategy on the number of surface FG that is ascribed to an interaction of the QDs with the carboxylic acid groups on the bead surface. These findings are of considerable relevance for applications of nanoparticle-encoded beads and safe-by-design concepts for nanomaterials.


Synthesis of CdSe/CdS-core/shell-QDs
The CdSe/CdS-QDs with a core/shell-architecture were prepared according to a previously described procedure 1 adapted from Carbone et al., Nightingale et al. and Chen et al.. [2][3][4] In the first step, CdSe cores with wurtzite structure were synthesised according to Carbone et al. 2 For this synthesis, 120 mg (0.93 mmol) CdO together with 560 mg (1.67 mmol) ODPA and 6 g (15.51 mmol) TOPO were degassed at 150 °C for 1 h. The mixture was then heated under argon flow to 300 °C. After the injection of 2 mL (4.48 mmol) of TOP, it was heated to 380 °C and, following a retention period of 10 min, 3.6 mL of a previously prepared TOP/Se solution (120 mg/3.6 mL) was swiftly injected. The temperature was allowed to rise to 380 °C again before the reaction was quenched by addition of 5 mL of ODE and cooled down to 70 °C in an air stream. During the cooldown period, 5 mL of toluene was added to prevent solidification. The resulting particles were precipitated by methanol/isopropanol (1:2), centrifuged at 6,000 rcf and redispersed in 2 mL of hexane.
A Cd(oleate)2 precursor solution was synthesised according to Nightingale et al. 3 For this synthesis, a mixture of 1.284 g (1 mmol) CdO, 12.94 mL (40.77 mmol) of oleic acid and 7.04 mL of ODE was degassed for 10 min at 100 °C. The dispersion was heated to 180 °C under argon flow and kept there for 60 min under vigorous stirring. To remove water as a side product, the mixture was cooled to 120 °C and degassed for 45 min. The 0.5 M Cd(oleate)2 solution was used as prepared for the next step.
The growth of the CdS surface passivation shell was performed according to an adapted synthesis by Chen et al. 4 For this, 100 nmol of the CdSe cores (60 -100 µL) were dispersed in 3 mL of ODE and OLA, respectively. The mixture was carefully degassed for 30 min at 90 °C. In the meantime, the S and Cd precursor solutions were prepared. For the desired shell thickness of 10 monolayers, 3.191 mL of Cd(oleate)2 and 286 µL of 1-octanethiol were diluted to a total volume of 7 mL with ODE, respectively. The flask was then heated under argon flow in two steps to 310 °C. When reaching 240 °C, the simultaneous injection of the previously prepared Cd(oleate)2 and 1octanethiol solutions via syringe pump (6 mL, 3 mL/h) was initiated. After two hours, 1 mL of oleic acid was injected, and the temperature was kept at 310 °C for another hour. Finally, the reaction mixture was cooled down to RT in an air flow, and the particles were precipitated by addition of acetone, centrifuged, and redispersed in hexane.

Preparation and 1 H-NMR spectrum of polyethylene glycol-block-poly(ε-caprolactone)
The block-copolymer polyethylene glycol-block-poly(ε-caprolactone) (PEG-b-PCL) was prepared according to a previously reported procedure 1 adapted from Meier et al.. 5 For this, 800 mg of poly(ethylene glycol) was added to a dry flask together with 1536 µL(14.53 mmol) of εcaprolactone. The mixture was placed in a preheated aluminium heating block and stirred for 5 min at 130 °C, followed by the addition of one drop of Sn(II) 2-ethylhexanoate as a catalyst and initiator. The mixture was then stirred at 130 °C for 3 h before it was rapidly cooled with an ice bath, leading to the precipitation of a white, solid product. The raw product was then recrystallized by first dissolving it in a small amount of dichloromethane, followed by precipitation with n-heptane. The such obtained block-copolymer was then filtered and washed several times with n-heptane before drying. Characterization of the synthesized PEG-b-PCL was done by nuclear magnetic resonance spectroscopy (solution 1 H-NMR) at RT with a 400 MHz JEOL JNM-ECX400 spectrometer (Free University Berlin). The sample was prepared by dissolving 6 mg of PEG-b-PCL in 700 µL of CDCl3.  Figure S2: a) TEM image and b) corresponding histogram of the particle size distribution of the CdSe/CdS semiconductor core/shell-quantum dots (QDs).

Electron microscopy of CdSe/CdS-QDs
The mean particle size of the QDs was calculated from TEM images to be 10.3 ± 1.2 nm. The PLQY was determined to be 58% in hexane, and the Cd concentration of the QD dispersion was determined by atomic absorption spectroscopy (AAS) to be 32.85 mg/mL. Figure S3: Histograms of the particle size distributions resulting from TEM micrographs of a) unloaded PSMPs, b) QD-loaded PSMPs prepared with QDs present during polymerization (route i.), and c) QD-loaded PSMPs prepared with a post-synthetic swelling procedure (route ii.). Figure S4: Full range FTIR spectra of both QD-loaded PSMPs and unloaded, unfunctionalized PSMPs with a change in carbonyl peak intensity, measured with two different PSMP concentrations. The slight offset of the spectra (baseline value below 0) is caused by the normalization procedure, the peak deviation at the right end of the spectra can be ascribed to impurities. Figure S5: Comparison of zeta potentials of synthesized (blue) and commercially available 2 µm PSMPs (red), loaded with RITC and NR in a postsynthetic swelling step.